PROJECT SUMMARY Basement membranes (BMs) are dense, sheet-like extracellular matrices that surround most animal tissues and provide mechanical strength and signaling cues that control growth, differentiation, and survival. Genetic and regulatory disruptions in BM components underlie numerous diseases, including cancer, fibrosis, and diabetes as well as eye, kidney, vasculature, and skin disorders. Despite the importance of BM to human health, there are currently no animal models that allow comprehensive real time visualization of BM components in vivo, which limits the understanding of fundamental properties of BMs and hinders development of therapies to treat BM disorders. The overall objective of this proposal is to create a complete toolkit of endogenously fluorescently tagged BM components in C. elegans with methodologies to advance our understanding of BM regulation and renewal in normal and disease states. These tagged proteins can be visualized in vivo because C. elegans is optically clear. C. elegans also has single genes encoding most major BM protein families and receptors and conditional knockdown approaches to disrupt their activity. Preliminary work has pioneered Cas9-mediated homologous recombination to insert the mNeonGreen fluorophore in-frame with 27 BM-associated genes, including all core matrix components, most matricellular proteins and all BM-associated receptors. These strains have been verified for full length protein expression, BM localization and viability. To complete the objective of developing a new BM toolkit animal model the following two specific aims will establish methods: (1) to quantify dynamic alterations of BM components and receptors in distinct tissues, and (2) to follow BM component turnover for the first time in vivo. Under Aim 1, experimental approaches will be developed to reveal BM component variation in different organs during development and aging, how BM composition variation is mediated by BM receptors, and how BMs adapt after loss of a key component (modeling human disease). In Aim 2, matrix components tagged with the photoconvertible fluorophore mEos2 will be made and fluorescent recovery after photobleaching (FRAP) and optical highlighting techniques will be developed to determine how BMs are renewed during normal maintenance and rapid growth. The proposed study will powerfully advance our understanding of BMs by providing the first model to dynamically track BM levels to understand how BMs are assembled, adapt in disease states, and change over time. Further, preliminary FRAP and photoconversion studies of BM components are already revealing that most matrix components are remarkably dynamic and some even flow along the BM. This unexpected dynamic nature implies that BM structural and signaling properties can be altered on the order of minutes?a profound change in our view of BM regulation. The proposed research is thus significant, as the tools and methods created by completion of this work will not only advance our understanding of BMs during normal and disease states, but also open a new field of study into the dynamic nature of BMs.